Calcium-independent phospholipases A2 (iPLA2s) constitute an important group of intracellular enzymes which function to hydrolyze esterified fatty acids from membrane phospholipids in response to agonist stimulation, changes in intracellular calcium ion homeostasis, and alterations in cellular energy requirements (for reviews, see 1-3). In early studies, we and others demonstrated that the majority of PLA2 activity in most non-circulating mammalian cell types including smooth muscle cells (4), pancreatic jβ-cells (5,6), cardiomyocytes (7,8), and hippocampal neurons (9) was calcium-independent and inhibited by racemic (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (rac-BEL). Based upon activity assays, calcium requirements, loss of arachidonylated phospholipid mass, and inhibition of iPLA2 by rac-BEL (R-BEL), a diverse array of cellular processes has been proposed to be regulated by iPLA2s, including arachidonic acid (AA) release (10-15), cellular proliferation (16), assembly of VLDL (17), putinergic receptor-stimulated kallikrein secretion (18), apoptosis (19), endothelial cell PAF synthesis (20), and induction of iNOS and nitric oxide production (21).
Three distinct subclasses of iPLA2 have been identified at the genetic level (with subsequent confirmation of iPLA2 catalytic activity by recombinant technologies) and have been designated iPLA2α, iPLA2β, and iPLA2γ, in order of their discovery (22-24). The iPLA2s have been categorized based upon their strict conservation of nucleotide-binding (GXGXXG) and lipase (GXSTG) consensus sequences (FIG. 1). Two of the iPLA2 subclasses, iPLA2β and iPLA2γ, have been cloned from mammalian cDNA libraries while the ortholog of iPLA2α (patatin), at the time of this writing (with 98.7% of tire human genome sequenced), has not been identified in mammals. Calcium-independent phospholipase A2β contains eight ankyrin-repeat domains which are believed to facilitate intracellular sorting (23, 25, 26) and a CaM-binding domain near the C-terminus which binds calcium-activated CaM and regulates enzyme activity (27) (FIG. 1). The binding of CaM to iPLA2β results in inhibition of iPLA2β activity which is reversible through removal of Ca+2 and subsequent dissociation of CaM from the C-terminus of iPLA2β (27, 28). In this paradigm, iPLA2β is regulated through alterations in cellular calcium ion homeostasis and becomes activated after dissociation from its complex with Ca+2/CaM when intracellular calcium stores are depleted by SERCA inhibitors, calcium-ionophores, or agonist stimulation (29, 30). In contrast, the recently identified iPLA2γ does not bind CaM and its mechanisms of regulation are unknown at present.
Studies of iPLA2 have utilized the mechanism-based suicide inhibitor rac-BEL as a pharmacologic tool to identify the type of intracellular phospholipase A2 involved in many diverse cellular processes. Since rac-BEL inhibits both iPLA2β and iPLA2γ at low microflora concentrations (24, 25, 31, 32), it is impossible to assign rac-BEL-mediated inhibition of AA release to iPLA2β or iPLA2γ activities. Accordingly, it became necessary to develop pharmacologic approaches which could discriminate between iPLA2β and iPLA2γ to facilitate identification of their biologic roles. In addition, it has been reported in the Journal (33, 34) that high concentrations of BEL (25 μM) partially inhibit the magnesium-dependent cytozoic phosphatidate phosphohydrolase, PAP-1, which converts phosphatidic acid to diacylglycerol (DAG). In those investigations, it was proposed that PAP-1 inhibition by BEL would prevent activation of protein kinase C leading to attenuated AA release. However, “rescue” experiments in which PKC was exogenously activated by phorbol esters or diacyl glycerol analogs after BEL treatment were not reported by the authors to address their hypothesis (33, 34).